The long term goal of this project is to elucidate the molecular basis of contraction and regulation in striated muscle. This field is now in a revolutionary phase due to advances in genetic, crystallographic, and cryo-electron microscopic techniques. Our approach is to use cryo-electron microscopy to define the molecular structures of the actin and myosin filaments and to capture the dynamic molecular events that generate and regulate contraction. By combining our observations with the atomic structures of actin and the myosin head, we are approaching a near-atomic description of contractile events. Our Specific Aims are: (1) To define the conformation, disposition and interactions of the myosin heads (crossbridges) that characterize the relaxed state of myosin filaments. (2) To determine, on the millisecond time-scale, the structural changes that occur in the crossbridges on the myosin filaments when they are activated by Ca2+ binding or by light chain phosphorylation. (3) To define the position of tropomyosin in the actin filaments of relaxed muscle and the changes in position that characterize the activated state, when troponin binds Ca2+. (4) To capture cross bridge motions during the crossbridge cycle in vitro. The use of cryo-electron microscopy is key to this proposal. Specimens are rapidly frozen, which preserves native filament structure and arrests transient molecular conformations, and are then observed in the unstained, frozen-hydrated state. Techniques will be used to generate rapid (millisecond time-scale) transitions in Ca2+ and ATP levels, allowing dynamic events of contraction to be captured. Three-dimensional structures of filaments will be computed by Fourier-based three-dimensional reconstruction methods, and molecular fitting will be used to relate our results to atomic structures. Preliminary data show that all of these goals are feasible. This project will provide data crucial to our understanding of the molecular mechanism of contraction and its regulation. It will help to illuminate general mechanisms of actin-myosin based cell motility in nonmuscle cells. And it will deepen our understanding of the structure of healthy muscle, which is essential to an understanding of structural defects that occur in diseased states.